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WO2019013392A1 - Anticorps monoclonal obtenu à partir d'un antigène spécifique reconnaissant spécifiquement une protéine en2 ou composition pour le diagnostic du cancer de la prostate le contenant - Google Patents

Anticorps monoclonal obtenu à partir d'un antigène spécifique reconnaissant spécifiquement une protéine en2 ou composition pour le diagnostic du cancer de la prostate le contenant Download PDF

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Publication number
WO2019013392A1
WO2019013392A1 PCT/KR2017/011645 KR2017011645W WO2019013392A1 WO 2019013392 A1 WO2019013392 A1 WO 2019013392A1 KR 2017011645 W KR2017011645 W KR 2017011645W WO 2019013392 A1 WO2019013392 A1 WO 2019013392A1
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Prior art keywords
protein
antibody
prostate cancer
antigen
monoclonal antibody
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Korean (ko)
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윤태종
조은이
장승희
김진현
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Moogene Medi Co Ltd
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Moogene Medi Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates

Definitions

  • the present invention relates to a monoclonal antibody obtained from a specific antigen which specifically recognizes an EN2 protein or a composition for diagnosing prostate cancer containing the antibody.
  • An antibody is an antibody that generally has an epitope that allows an external substance (a substance with a certain size and condition) (antigen) to be injected into an organism and an organism capable of specifically recognizing an external substance through a humoral immune response Antibodies are formed and they are taken from the blood and separated from the serum. The antibody thus formed is a polyclonal antibody having several antigen recognition sites.
  • the antigen is present in the B cell as a small epitope by various antigen presenting cells (APC) and activates the B cell.
  • APC antigen presenting cells
  • the genes that make antibodies capable of reacting with the antigen are rearranged, and the remaining unnecessary genes are removed, so that only one antibody is produced in a large amount, and the plasma cells ). Since the antigen generally has several epitopes, different kinds of plasma cells are different, and thus various antibodies are produced.
  • a total of several kinds of antibodies secreted from various kinds of plasma cells that is, genes that make antibodies
  • are referred to as 'polyclonal antibodies' and only one type of antibody produced in one kind of plasma cells Clone antibody ".
  • the diagnostic market using antibodies has been rapidly increasing since 1980 and it has been used for the development and diagnosis of highly efficient diagnostic kits because it can detect very small amount of protein specifically expressed by disease or symptom .
  • it is essential to develop antibodies with both specificity and sensitivity to proteins (antigens) expressed by diseases and symptoms.
  • the efficiency of the antibody can be maximized by some consideration.
  • the antigen considering the nature of the antigen and the quality of the antigen as a function of the steric structure in the sequence after modification after translation, it is considered to be selected. Particularly when the whole protein is not used as an antigen but when it is used as an antigen in the form of some peptides, it should be selected especially.
  • the antibody should be prepared according to the purpose. In the case of polyclonal antibody, it is easy to produce, it is easy to detect the antigen due to various epitopes, and it has a wide selection of immune animals according to the antigen. However, antibody specificity drops and it is not easy to mass-produce the same activity.
  • Monoclonal antibodies have the advantage of producing antibodies in culture supernatant, which can produce large quantities of antibodies having the same specificity of activity. However, it takes a long time to produce antibodies, limited immune animals, It is not suitable. Therefore, monoclonal antibodies can be produced when mass production of an antibody having the same activity is required after confirming specificity of an antigen through production of polyclonal antibodies.
  • a prostate is a biliary-sized male reproductive organs beneath the bladder, in front of the rectum, that produces and stores part of the semen.
  • the upper part is fixed to the bladder neck, ie, the part of the bladder to the urethra, adjacent to the anterior tibial prostate ligament, and the lower part is fixed by the genitourinary diaphragm.
  • Most of the cancer that occurs in the prostate gland is adenocarcinoma (cancer of the gland cell) that occurs in the prostate gland.
  • the types can be classified according to the degree of differentiation of the tumor tissue and the characteristics of the cells. Prostate cancer is one of the most common urological tumors worldwide.
  • human prostate cancer appears to have a tendency to metastasize to bone and is known to increase the mortality rate of patients by inevitably progressing from an androgen-dependent state to an androgen-resistant state. Furthermore, about 25% of men who have received prostate cancer treatment are those that require additional treatment due to recurrence of disease, which is currently the second leading cause of cancer deaths in the United States in the United States. .
  • PSA prostate-specific antigen
  • a very high serum PSA level can be used as a standard diagnostic tool for effective prostate cancer, while a weak PSA serum level of 2-10 ng / ml can lead to a definite diagnosis of prostate cancer I can not.
  • the PSA assay for diagnosing prostate cancer is problematic in terms of detection specificity because the serum PSA in such a weakly rising state may be derived from a non-tumor disease such as benign prostatic hyperplasia (BPH), prostatitis or other physical trauma . Therefore, the diagnosis of prostate cancer using a new biomarker has become a major subject and research has been conducted (Korean Patent Laid-Open No. 10-2009-0111307).
  • EN2 (Engrailed-2) has been used as a biomarker to diagnose prostate cancer.
  • EN2 is a biomarker capable of solving the problem of detection specificity of PSA (prostate specific antigen) (bio-marker).
  • the EN2 protein acts as a transcription factor in the cell and is overexpressed only in the prostate cancer cell, resulting in a DNA transcriptional regulatory disorder.
  • EN2 expression increases in prostate cancer cells, the amount of EN2 protein excreted in the urine is also increased, which is suitable for in vitro analysis.
  • US Pat. Nos. 8460882, 2012-532621 or USP 8722643 disclose techniques relating to a diagnostic composition for recognizing EN2 protein, and Korean Patent Laid-Open No. 10-2016-0077788 Techniques relating to DNA plasmids that specifically bind EN2 are disclosed.
  • the inventors of the present invention studied a composition relating to prostate cancer diagnosis, and produced a monoclonal antibody which can more effectively diagnose EN2 protein.
  • the present invention can be completed by manufacturing a prostate cancer diagnostic composition using the antibody.
  • Patent Document 1 U.S. Patent No. 8460882 (entitled “Cancer biomarkers”, Applicant: The University of Surrey, Registered on March 31, 2013)
  • Patent Document 4 Japanese Laid-Open Patent Publication No. 2012-532621 (Title of the invention: Therapeutic peptides, polypeptides and nucleic acid sequences, Applicant: The University of Surrey,
  • Patent Literature 5 Korean Patent Laid-Open No. 10-2009-0111307 (entitled DNA Platamer specifically binding to EN2 and its use, Applicant: Industry-Academic Collaboration Foundation, POSTECH, Publication date: 2016.07.04)
  • Patent Document 6 Korean Patent Laid-Open No. 10-2016-0077788 (the title of the invention: use of the prostate-specific transcript and its use in the diagnosis and diagnosis of prostate cancer, applicant: Exxon Heat Practicals SA, : October 26, 2009.)
  • the present invention provides a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 or 3; And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, 5, 6 or 7.
  • the antibody is also characterized by recognizing an epitope consisting of the amino acid sequence of SEQ ID NO: 10.
  • the monoclonal antibody of the present invention is characterized by recognizing EN2 (Engrailed-2, Accession No. NP - 001018.2) protein or its recombinant protein consisting of the amino acid sequence of SEQ ID NO: 1.
  • the antibody is an immunoglobulin G type.
  • the present invention can also provide a diagnostic composition for diagnosing the presence or absence of EN2 (Engrailed-2) protein comprising said monoclonal antibody or a prostate cancer diagnostic agent.
  • the present invention also relates to a hybridoma cell line that is a Accession No. KCLRF-BP-00405 and provides a monoclonal antibody produced by said hybridoma cell line.
  • the EN2 protein, the recombinant protein thereof, the fragment of the EN2 protein and the amino acid sequence information contained in the antibody recognizing the EN2 protein used in the present invention are as follows.
  • the protein consisting of the amino acid sequence of SEQ ID NO: 1 below relates to the EN2 protein expressed in the mammal, especially the human body.
  • SEQ ID NO: 1 NCBI Reference Sequence Accession No. NP_001418.2
  • the amino acid sequences of SEQ ID NOS: 2 to 7 below are amino acid sequences constituting the monoclonal antibodies of the present invention.
  • the antibody comprising the variable heavy chain or variable light chain sequence of SEQ ID NOS: 2 to 7 below is a monoclonal cell obtained by fusion of an immunocompetent lymphocyte with an EN2 peptide consisting of the amino acid sequence of SEQ ID NO: 9 and myeloma cells (Accession number KCLRF-BP-00405).
  • EN2 peptide consisting of the amino acid sequence of SEQ ID NO: 9 and myeloma cells (Accession number KCLRF-BP-00405).
  • the underlined part of each sequence is the sequence of CDR3.
  • SEQ ID NO: 2 Variable heavy chain amino acid sequence of EN2 monoclonal antibody
  • SEQ ID NO: 3 Variable heavy chain amino acid sequence of EN2 monoclonal antibody
  • SEQ ID NO: 4 Variable light chain amino acid sequence of EN2 monoclonal antibody
  • SEQ ID NO: 5 Variable light chain amino acid sequence of EN2 monoclonal antibody
  • SEQ ID NO: 6 Variable light chain amino acid sequence of EN2 monoclonal antibody (antigen: EN2 peptide consisting of amino acid sequence of SEQ ID NO: 15)
  • SEQ ID NO: 7 Variable light chain amino acid sequence of EN2 monoclonal antibody (antigen: EN2 peptide consisting of amino acid sequence of SEQ ID NO: 15)
  • the amino acid sequence of SEQ ID NO: 8 below is the amino acid sequence constituting the EN2 recombinant protein used for the identification of the monoclonal antibody in the present invention.
  • the EN2 recombinant protein consisting of the amino acid sequence of SEQ ID NO: 8 below contains the T7 tag (b) to enhance the expression efficiency of the EN2 protein (Accession No. NP_001418.2) and contains the His tag (a) .
  • E. coli expression vector was used for gene recombination. For this purpose, restriction enzyme site - (c) is used.
  • SEQ ID NO: 8 Recombinant EN2 protein
  • SEQ ID NO: 9 fragment (peptide) of EN2 protein for producing the antibody of the present invention
  • the monoclonal antibody of the present invention recognizes the EN2 protein.
  • the EN2 protein may be extracorporeally extracted from a mammalian body, or may be a recombinant EN2 protein.
  • the present invention can confirm or quantify the presence or absence of EN2 protein in the body of a patient with prostate cancer. That is, the present invention can provide a method of diagnosing the presence or absence of EN2 protein by specifically recognizing EN2 protein using the above monoclonal antibody. At this time, the presence or absence of the EN2 protein can be confirmed by any conventional experimental method used for the presence or the quantification of the existing protein.
  • the present invention also provides an EN2 monoclonal antibody composition which specifically recognizes an EN2 protein or a diagnostic agent containing the EN2 monoclonal antibody composition.
  • the diagnostic agent may include a labeled secondary antibody, a chromophore, an enzyme conjugated with the antibody, and other substances capable of binding to the substrate or antibody.
  • the diagnostic agent of the present invention may include a recombinant EN2 protein capable of quantifying the EN2 protein contained in the body of the subject to be diagnosed.
  • an antibody refers to a specific protein molecule directed against an antigenic site.
  • the antibody of the present invention may preferably comprise a monoclonal antibody that specifically binds to the EN2 protein.
  • the monoclonal antibodies of the present invention can be prepared using the hybridoma method (Kohler G. and Milstein C.) or the phage antibody library (Clackson et al. Marks et al.) Technique, as is well known in the art to which the present invention pertains. .
  • the hybridoma method cells of an immunologically appropriate host animal such as a mouse and a cancer or myeloma cell line can be used. After these two types of cells are fused with each other by a method using polyethylene glycol or the like, that is, a method widely known in the technical field to which the present invention belongs, the antibody producing cells can be proliferated by a standard tissue culture method .
  • a uniform cell population is obtained by subcloning by a limited dilution technique, and then a hybridoma capable of producing an antibody specific for the recombinant protein of the present invention can be produced in vitro or in vivo Lt; / RTI >
  • the above-described phage antibody library method comprises the steps of obtaining an antibody gene for a recombinant protein of the present invention and expressing it in the form of a fusion protein on the surface of a phage to prepare an antibody library in vitro, A monoclonal antibody that binds to a protein can be isolated and produced.
  • the antibodies produced by the above methods can be separated by methods such as centrifugation, electrophoresis, dialysis, ion exchange chromatography, affinity chromatography and the like.
  • the present invention provides a method for producing a monoclonal antibody using an immunogenic fragment peptide (EN2 peptide) of EN2 protein.
  • the EN2 monoclonal antibody of the present invention can be prepared by injecting an animal with an immunogenic fragment peptide of EN2 protein as an antigen and then isolating the spleen tissue and using fusion technology with myeloma cells.
  • the animal may be any animal host such as a mouse or a rat.
  • step 1 preparing an immunogenic fragment peptide of the EN2 protein as an antigen for producing an antibody
  • Step 2 mixing and emulsifying the antigen and the adjuvant
  • Step 4 After 7 to 20 days from the administration of the antigen, serum is separated from the animal, the activity of the antibody is confirmed, and then the spleen of the animal is separated.
  • Step 5 A lymphocyte is isolated from the spleen, mixed with myeloma cells to induce cell fusion, and monoclonal cells producing an antibody having a high antigen-antibody reaction to EN2 protein or EN2 recombinant protein among fused cells Selecting;
  • Step 6 culturing the selected monoclonal cells to obtain immunoglobulin from the culture medium
  • Step 7 separating and purifying the immunoglobulin
  • the antibody may comprise a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains.
  • a functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab) 2, F (ab) 2, Fv and the like.
  • any animal can be used as an animal which can cause an immune response, but it may be preferable to use a mouse most easily used for production of a monoclonal antibody.
  • the immunoglobulin of the sixth step may be any type, but preferably it may be immunoglobulin G.
  • the present invention also provides a method of diagnosing prostate cancer using an EN2 protein or a recombinant protein thereof or an antibody specifically recognizing the same.
  • Step 2 contacting the sperm protein of Step 1 with the antibody of the present invention to form an antigen-antibody complex
  • Step 3 Detecting and analyzing the antigen-antibody complex produced in Step 2 in a quantitative manner.
  • the sample of the first stage may be extracted from a subject to be confirmed whether or not prostate cancer has occurred or progressed, and preferably may be tissue, cells, etc. of a mammal (human or other animal) May be urine, blood, plasma, serum, liver cells, and the like.
  • the step of separating the body protein in the above step 1 can be carried out using a known process, and the amount of the body protein can be measured by various methods known to those skilled in the art.
  • the amount of the antigen-antibody complex formed in the control group and the amount of the antigen-antibody complex formed in the subject in which the presence or absence of the prostate cancer is confirmed can be compared through the above-described analysis method, and the presence or absence of the prostate cancer Of prostate cancer can be directly diagnosed by judging the amount of EN2 protein expression.
  • the EN2 content of the sample to be inspected for the presence or absence of progression of prostate cancer in step 3 can be confirmed through a standard value of the concentration of the recombinant EN2 protein used in the present invention.
  • the amount of EN2 protein expressed in the presence or absence of progression of prostate cancer is determined by whether it corresponds to 3.1 to 65.4 nM (Sci.Rep. 2013; 3: 2059), which is known as urine EN2 protein concentration range in patients with prostate cancer .
  • the formation amount of the antigen-antibody complex can be quantitatively measured through the signal size of the detection label.
  • the detection label may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule and a radioactive isotope, but is not limited to the substance described above.
  • the enzyme When the enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,?
  • -D-galactosidase urease, peroxidase or alkaline phosphatase , Acetylcholinesterase, glucose oxidase, and hexokinase
  • the fluorescent material include fluorescein, phycocyanin, fluorescamine, and the like, but the material is not limited to those described above.
  • the ligand include, but are not limited to, biotin derivatives and the like.
  • the luminescent material includes, but is not limited to, luciferin.
  • the fine particles include, but are not limited to, colloid, gold, and the like.
  • the redox molecules include, but are not limited to, quinone, 1,4-benzoquinone, and hydroquinone.
  • radioisotopes include, but are not limited to, 3 H, 14 C, and the like.
  • the diagnostic method can be performed by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, Immunocytochemistry, immunoprecipitation, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, and the like.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • RIA radioimmunodiffusion
  • ouchterlony immunodiffusion ouchterlony immunodiffusion
  • rocket immunoelectrophoresis Immunocytochemistry, immunoprecipitation, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, and the like.
  • FACS fluorescence activated cell sorter
  • the subject to be diagnosed using the antibody of the present invention can be any subject whose EN2 protein is produced in the body, and it is possible to diagnose all mammals including human.
  • the mammal may be any kind of mammal such as a person, a dog, a cat, a rabbit, a cattle, a goat, a pig, and a horse.
  • the present invention relates to an EN2 monoclonal antibody that specifically recognizes an EN2 protein.
  • the EN2 monoclonal antibody can be used as a diagnostic agent composition for prostate cancer because it can detect the presence or absence of EN2 protein present in the body and is significantly superior in detection sensitivity to a conventional EN2 protein antibody.
  • diagnosis efficiency is higher than that of prostate-specific antigen (PSA) detection in blood, which is a conventional method of diagnosing prostate cancer.
  • PSA prostate-specific antigen
  • EN2 monoclonal antibody having the amino acid sequence as disclosed in the present invention or EN2 monoclonal antibody prepared from the hybridoma cells of the present invention Can be said to be a novel composition completely different in technical characteristics from those disclosed in the prior art.
  • Fig. 1 (A) is a schematic representation of the cloning of an EN2 (homeobox protein engrailed-2) protein in a large amount by using an E. coli expression vector in Example 1-1, and B is a large- And purified and purified recombinant EN2 protein by SDS-PAGE.
  • FIG. 2 (A) is the HPLC result obtained by synthesizing and purifying peptides expected to have high antigenicity of EN2 (homeobox protein engrailed-2) in Example 1-2, and B shows the result of mass spectrum of this EN2 peptide Results.
  • FIG. 3 shows the results of immunization of the polyclonal antibody (FIG. 3A) obtained in Example 2-2 with the EN2 peptide prepared in Example 1-2 as an antigen and the fusion of lymphocytes and myeloma cells of the immunological animal producing the polyclonal antibody (FIG. 3B) obtained from the hybridoma cells of Example 3-3 obtained through the above-described method of the present invention.
  • the polyclonal antibody is represented by Anti-EN2 (Poly)
  • the monoclonal antibody of the present invention is represented by Anti-EN2 (Mono).
  • Figure 4A shows the selective detection of prostate cancer cell line PC3 and LNCaP expressing EN2 protein for the EN2 monoclonal antibody (Anti-EN2 (Mono)) of the present invention and the comparative group polyclonal antibody (Anti-EN2 blot experiment, and B is a numerical result.
  • FIG. 5A shows the quantifiable concentration of the antigen-detectable recombinant EN2 protein against the EN2 monoclonal antibody (Anti-EN2 (Mono)) of the present invention and the comparative group polyclonal antibody (Anti-EN2 , And B is the result of quantification.
  • FIG. 7A shows the result of ELISA (EN2 protein quantification standard line) for the selective detection of EN2 protein of the EN2 monoclonal antibody (Anti-EN2 (Mono)) of the present invention and B using the result of A ELISA shows the results of quantitation of EN2 protein in a sample (urine) of actual benign prostatic hyperplasia (P1), prostatitis patient (P2) or prostate cancer patient (P3 ⁇ P7).
  • EN2 protein quantification standard line for the selective detection of EN2 protein of the EN2 monoclonal antibody (Anti-EN2 (Mono) of the present invention
  • B using the result of A ELISA shows the results of quantitation of EN2 protein in a sample (urine) of actual benign prostatic hyperplasia (P1), prostatitis patient (P2) or prostate cancer patient (P3 ⁇ P7).
  • FIG. 8A shows the use of the EN2 monoclonal antibody (Anti-EN2 (Mono)) of the present invention to determine whether the sample (urine) of the actual patients P1 to P7 can distinguish between prostate cancer and prostatitis or benign prostatic hyperplasia western blot method, and B is the prostate-specific antigen (PSA) concentration of these patients.
  • EN2 monoclonal antibody Anti-EN2 (Mono)
  • PSA prostate-specific antigen
  • FIG. 9 shows the results of analyzing the amino acid sequences of the light chain and the heavy chain of the monoclonal antibody prepared in the present invention.
  • Fig. 10 shows the epitope sequence predicted through epitope mapping using the monoclonal antibody of the present invention.
  • Example 1-1 Recombinant EN2 protein cloning, expression, purification
  • FIG. 1 (a) illustrates the pET28b / EN2 plasmid.
  • the pET28b / EN2 plasmid was transformed into E. coli (BL21 / DE3) and overexpressed EN2 protein at 0.1 mM IPTG and 37 °C. Overexpressed E.
  • the eluate buffer (20 mM Tris-Cl at pH 8.0, 300 mM NaCl, 300 mM imidazole, 1x protease inhibitor cocktail)
  • the imidazole was removed by dialysis (cut off 10K) in a storage buffer (50 mM Tris-HCl pH 8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% glycerol) Bicinchoninic acid) and the absorbance at 280 nm.
  • each of the peptides was scored with an antigenic index and scored from 1 to 5 And a score of 3.8 or higher was selected.
  • the antigenic index scored the potential as an antigen due to structural influences and stereostructures according to post-translational modification of nucleotides to proteins.
  • the regions where the hydrophobic residues are distributed are excluded from the selection because the external exposure is blocked in the folding of the proteins.
  • the sequence of the peptides selected according to the above criteria is the highest 10 The peptide with the least hydrophobic residue was selected.
  • a carrier protein KLH (keyhole limpet hemocyanin) was cross-linked using a cross-linker.
  • peptides, carrier proteins, and cross-linking agents were dissolved at a weight ratio of 1: 1: 1 and sufficiently reacted at room temperature for 2 hours or more.
  • EDC 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride
  • 0.1 M MES 2- [N-morpholino] ethane sulfonic acid (Where the buffer serves to induce the hydrolysis of the amine group).
  • This EN2 peptide conjugated with the transporter protein was then used as an antigen for preparing the antibody of the present invention.
  • Example 1-2 In order to administer the antigen to the immune animal, it is necessary to bond with the adjuvants so that the antigen having a high solubility in the living body can stay for a long time, thereby preparing a composition in which the adjuvant and the antigen are emulsified.
  • the antigens prepared in Example 1-2 were administered four times in total.
  • the first administration antigen was mixed with Freund's complete adjuvant (FCA) containing killed mycobacterium And Freund's incomplete adjuvant (FIA), which excluded dead mycobacterium, was prepared by mixing the remaining three antigens for administration.
  • FCA Freund's complete adjuvant
  • FIA killed mycobacterium And Freund's incomplete adjuvant
  • the antigen and the adjuvant are mixed using a probe sonication method, and the mixture is kept at 4 ° C so that heat is not applied to the antigen during the mixing.
  • Antigen and adjuvant were mixed in a 1: 1 volume.
  • Example 2 Thereafter, in order to prepare a monoclonal antibody having a good reactivity to the EN2 protein, a process for screening a hybridoma cell that produces a monoclonal antibody from the lymphocyte of the animal is selected and an immunized animal that produces a serum with good antibody reaction is selected.
  • a process for screening a hybridoma cell that produces a monoclonal antibody from the lymphocyte of the animal is selected and an immunized animal that produces a serum with good antibody reaction is selected.
  • Example 2-1 Selection of immune animals
  • a mouse (BALB / C) was used to induce an immune response to human EN2 protein sequence. Immunoreactivity was induced in two 5-week-old females against the antigen (peptide mixed with the adjuvant of Example 1-3) in consideration of the individual specificity, since the degree of immune response may vary depending on individuals. The same kind of mice were used as immune animals used for the production of monoclonal antibodies.
  • Example 2-2 Induce the immune response and check the activity of the antibody
  • the first antigen administration 100 ⁇ l of the emulsion was injected into the plantar bones of experimental mice (BALB / C) to induce an immune response. Two weeks after the first injection, 100 ⁇ l of the same antigen was administered to the plantar feet of the experimental mice to induce an immune response. Re - injection was performed up to 4 times at intervals of 1 week. After immunization, a small amount of serum was collected and the activity of polyclonal antibody in serum was confirmed by ELISA test.
  • ELISA confirmation antigen (EN2 recombinant protein) was diluted to 5 ⁇ g / ml in coating buffer, and 50 ⁇ l per well was coated on 96 well plate and reacted overnight at 4 °C. The following day, the cells were blocked with 2% skim milk for 1 hour, diluted to the primary antibody, and reacted at 37 ° C for 2 hours. The reaction plate was washed three times with TBS-T, then diluted with 1: 10,000 ratio of goat anti-mouse IgG conjugated with HRP, and 50 ⁇ l of each was diluted in the plate well and reacted at 37 ° C for 1 hour.
  • Example 3-1 Fusion - Preparation of hybridoma cells
  • Lymphocytes were isolated from the immunized animals producing antibodies with high titers based on the titer in the serum of the immunized mice identified in Example 2, and cell fusion was performed.
  • mice lymph nodes were removed without damage and washed with DMEM (Dulbecco / Vogt modified Eagle's minimal essential medium) medium. Lymphocytes were separated and dispersed in DMEM medium in a 60 mm dish into single cells. / 0 Ag 14 - ATCC # CRL-1581) and lymphocytes were mixed at a ratio of 1: 1 to the number of cells.
  • DMEM Dulbecco / Vogt modified Eagle's minimal essential medium
  • ECF buffer 0.3M mannitol, 0.1mM calcium chloride, 0.1mM magnesium chloride
  • ECF buffer 0.3M mannitol, 0.1mM calcium chloride, 0.1mM magnesium chloride
  • the cell fusion was induced by applying an electrode of about 1.3 volts in an electrode container.
  • Cells were seeded at a density of 200 ⁇ l per well in a 96-well plate (2.5 ⁇ 10 7 cells / plate) and cultured in a CO 2 incubator. Fusion After 5 days, 50 ⁇ l of HAT media (hypoxanthine-aminopterin-thymidine medium) was added to each well. After 12 days of fusion, colony was identified and the reaction with EN2 recombinant protein (ELISA confirmation antigen) was analyzed by ELISA (Asb. 450 nm) method. The procedure was the same as in Example 2.
  • Example 3 Cloning : Hybridoma selection to produce an antibody having excellent antigen-antibody reactivity
  • the positive selection criterion of the ELISA performed in Example 3-1 is an OD value of 1.0 or more (OD value of 1 or less means that the affinity of the actual monoclonal antibody is not good after the next cloning step , And non specific binding also occurs in some OD values, so it is better to select a minimum value of 1 or more.) Since most of the values are more than 0.5 in the confirmation result, more than 0.5 positive And monoclonal cells were selected.
  • cloning was performed to select monoclonal cells from selected wells based on the ELISA OD value after cell fusion.
  • the cells were transferred to 24 wells and subjected to ELISA test.
  • the cells were spun down and dispersed with HT media. Cell counting was performed so that 150 cells per 96-well plate were dispensed.
  • ELISA test (the same method as in Example 3-1) was performed, and a second colony was selected by selecting an ELISA test result with a low colony and a high OD value (ELISA positive selection criteria The same as in Example 3-1). Thereafter, further cloning was continued, and the respective cloning procedures were the same as in Example 3-1. From the fourth round of cloning, complete media (DMEM, 10% FBS [Fetal bovine serum] + 1% PS [Penicillin-Streptomycin]) was used.
  • DMEM 10% FBS [Fetal bovine serum] + 1% PS [Penicillin-Streptomycin]
  • Example 4-1 Collection of monoclonal cell culture media
  • Example 3-3 The cells obtained in Example 3-3 were dissolved at 37 ° C and cultured in DMEM / 10% FBS / 1% PS medium for 10 days in a 5% CO 2 humidified incubator. Subsequently, subculture was carried out at intervals of two days to collect the culture medium Respectively.
  • the culture medium contains various proteins including FBS, it was decided to purify only immunoglobulin G (IgG) in order to increase the activity and specificity of the antibody.
  • IgG immunoglobulin G
  • the culture medium was bound to packed resin (Protein G Sepharose 4 Fast Flow-GE Health care combined with protein G binding specifically to mouse IgG) and washed with a buffer of 2-3 times bead volume
  • Elution buffer used pH 2-3 buffer to break the specific binding between protein G and IgG.
  • Tris pH 9
  • Elution was divided into 10 fractions of 1 ml each after 3 ml Elution per resin. Using 10% SDS-PAGE, the fractions were mixed with 9 ⁇ l of fraction and 3 ⁇ l of 4x sample buffer (containing reducing agent) After boiling for minutes, they were loaded.
  • A Polyclonal Clone
  • B is an antibody fragment obtained from a monoclonal cell (hybridoma) producing the antibody of the present invention .
  • the antibody of FIG. 3A was used as a comparative group in the subsequent experiments.
  • the Polyclonal Clone antibody was labeled as Anti-EN2 (Poly), and the monoclonal antibody of the present invention was labeled Anti-EN2 (Mono).
  • Each antibody (IgG) finally obtained was diluted in PBS / 0.2% sodium azide / 20% glycerol buffer, dispensed, and stored at -80 ° C.
  • Example 5 Evaluation of EN2 protein detection ability of EN2 monoclonal antibody in prostate cancer cell line >
  • EN2 expression is high prostate cancer cell line PC3 (® CRL-1435 TM) and LNCaP (® CRL-1740 TM) the purchased from ATCC (American Type Culture Collection, Rockville, MD, USA) and this 37 °C, 5% CO 2 (FBS) and 1% penicillin / streptomycin (P / S) were added to RPMI-1640 (PC3) or RPMI-1640 (LNCaP) medium containing 25 mM HEPES in a humidified incubator.
  • the lysed proteins were suspended in 40 ⁇ g / well and 20 ⁇ g / well 20 ⁇ l of each well was added to each well and separated by 4-15% SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). PAGE were transferred to a PVDF membrane and each EN2 antibody (6.6 nM) (Anti-EN2 (Poly), Anti-EN2 (Mono)) was added to 5% skim milk / TBS-T (Tris-saline + Tween20) The HRP-conjugated anti-mouse antibody was diluted with 5% skim milk / TBS (1: 2000) at 4 ° C.
  • FIG. 4 where FIG. 4A is a band image obtained by performing Western blotting, and FIG. 4B is a graph showing the result of the Western blotting.
  • anti-EN2 (mono) of the present invention is superior to EN2 protein in all two prostate cell lines.
  • anti-EN2 (poly) It is confirmed that the antigen-antibody reaction does not appear. This may be a disadvantage in the process of producing polyclonal antibodies because the non-reactive antibodies may be produced according to the degree of antigen-antibody reaction in the blood depending on the immune animal.
  • the use of polyclonal antibodies may not be useful for quantitative analysis of EN2 proteins, since antigen-antibody activity may vary for each immune animal.
  • a monoclonal antibody is used as in the present invention, it is very effective in that a large quantity of antibodies of the same quality can be produced through hybridoma cells.
  • EN2 protein is detected at a concentration of at least 1 ng / 20 ⁇ l (0.05 ng / ⁇ l) (1.2 nM) in Anti-EN2 (mono) ,
  • the antigen-antibody reactivity is excellent and the protein-detecting ability of the monoclonal antibody is excellent.
  • EN2 protein can be detected sensitively even when applied to actual urine samples (urine EN2 protein concentration range of prostate cancer patients: 3.1 to 65.4 nM: Sci.Rep. 2013; 3: 2059).
  • the intracellular EN2 protein of prostate cancer cell lines LNCap and PC3 was detected by immunofluorescence staining.
  • Anti-EN2 (Mono) and anti-EN2 (Poly) were used as the primary antibodies and anti-EN2 (Poly) as the secondary antibodies.
  • Anti-mouse IgG / 594 was used as the secondary antibodies. Binding capacity was analyzed by Confocal Laser Scanning Microscope. The fluorescence staining photographs thereof are shown in Fig.
  • the antibody of the present invention was used to confirm whether the classification of prostate disease patients was confirmed.
  • Monoclonal cells producing anti-EN2 (mono) antibody were provided to Y biologics (Daejeon), and amino acid sequence analysis of the light and heavy chains of the monoclonal antibody was performed.
  • the results are shown in Fig.
  • SEQ ID NOS: 2 and 3 represent the variable heavy chain amino acid sequence of the antibody
  • SEQ ID NOS: 4 to 7 represent the variable light chain amino acid sequence of the antibody, representing the amino acid sequence of the light chain and the heavy chain.

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Abstract

La présente invention concerne un anticorps monoclonal anti-EN2 reconnaissant spécifiquement la protéine EN2. L'anticorps monoclonal anti-EN2 permet de confirmer la présence ou l'absence de protéine EN2 dans un corps et de la quantifier, et a une sensibilité de détection significativement excellente par comparaison avec des anticorps anti-protéine EN2 existants, l'anticorps monoclonal anti-EN2 peut être utilisé efficacement en tant que composition pour le diagnostic du cancer de la prostate. Lors du diagnostic du cancer de la prostate à l'aide de l'anticorps de la présente invention, il a été confirmé qu'il y a une efficacité de diagnostic plus élevée que celle d'un résultat de détection d'antigène spécifique de la prostate (PSA) dans le sang, qui est une méthode de diagnostic existante pour le cancer de la prostate.
PCT/KR2017/011645 2017-07-14 2017-10-20 Anticorps monoclonal obtenu à partir d'un antigène spécifique reconnaissant spécifiquement une protéine en2 ou composition pour le diagnostic du cancer de la prostate le contenant Ceased WO2019013392A1 (fr)

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WO2022129976A1 (fr) * 2020-12-17 2022-06-23 Sorbonne Universite Polypeptides se liant sélectivement à des glycosaminoglycanes de type héparine ou sulfate d'héparane et polypeptides de pénétration cellulaire comprenant ceux-ci

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KR101777254B1 (ko) * 2017-07-14 2017-09-13 주식회사 무진메디 En2 단백질을 특이적으로 인식하는 특정 항원으로부터 얻어진 단클론 항체 또는 이를 함유하는 전립선암 진단용 조성물

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112940115A (zh) * 2019-12-10 2021-06-11 北京格根生物科技有限公司 一种抗Engrailed-2蛋白单克隆抗体及其应用
CN112940115B (zh) * 2019-12-10 2022-07-12 北京格根生物科技有限公司 一种抗Engrailed-2蛋白单克隆抗体及其应用
WO2022129976A1 (fr) * 2020-12-17 2022-06-23 Sorbonne Universite Polypeptides se liant sélectivement à des glycosaminoglycanes de type héparine ou sulfate d'héparane et polypeptides de pénétration cellulaire comprenant ceux-ci
EP4263569A1 (fr) * 2020-12-17 2023-10-25 Sorbonne Universite Polypeptides se liant sélectivement à des glycosaminoglycanes de type héparine ou sulfate d'héparane et polypeptides de pénétration cellulaire comprenant ceux-ci

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